Increasing ornithine accumulation to increase high mannose glycoform content of recombinant proteins

ABSTRACT

The present invention relates to a method for manipulating the high mannose glycoform content of recombinant glycoproteins by regulating ornithine metabolism during cell culture.

This application is a divisional of pending U.S. application Ser. No.15/111,470, filed on Jul. 13, 2016; which is a 371 of InternationalPatent Application No. PCT/US14/069378, filed Dec. 9, 2014; which claimsthe benefit of U.S. Provisional Application No. 61/926,481 filed Jan.13, 2014, each of which is incorporated by reference herein.

BACKGROUND OF INVENTION

A variety of post-translational modifications including methylation,sulfation, phosphorylation, lipid addition and glycosylation areperformed by higher eukaryotes. Many of the secreted proteins, membraneproteins and proteins targeted to vesicles or certain intracellularorganelles are known to be glycosylated. Glycosylation, the covalentattachment of sugar moieties to specific amino acids, is one of the mostcommon, yet important post-translational modifications for recombinantproteins. Protein glycosylation has multiple functions in the cellincluding its essential role in protein folding and quality control,molecular trafficking and sorting, and cell surface receptorinteraction.

N-linked glycosylation involves addition of oligosaccharides to anasparagine residue found in certain recognition sequences in proteins(e.g., Asn-X-Ser/Thr). N-linked glycoproteins contain standard branchedstructures which are composed of mannose (Man), galactose,N-acetylglucosamine and neuramic acids. High-mannose oligosaccharidestypically include two N-acetylglucosamines with multiple mannoseresidues (5 or more). Glycoproteins produced in mammalian cell culturemay contain varied levels of these high mannose (HM or HMN) glycoformssuch as Mannose5 (Man5), Mannose6 (Man6), Mannose7 (Man7), Mannose8(Man8) and Mannose9 (Man9).

While the glycoforms of a recombinant glycoprotein expressed by Chinesehamster ovary (CHO) host cell are largely determined by intrinsicgenetic factors, high mannose glycoform content can also be affected bycell culture conditions (Pacis, et al., (2011) Biotechnol Bioeng 108,2348-2358).

Glycosylation can affect therapeutic efficacy of recombinant proteindrugs. The influence of glycosylation on bioactivity, pharmacokinetics,immunogenicity, solubility and in vivo clearance of therapeuticglycoproteins have made monitoring and control of glycosylation acritical parameter for biopharmaceutical manufacturing. The high mannoseglycoform content of therapeutic proteins is a critical qualityattribute that has been found to affect pharmacokinetic properties ofcertain therapeutic antibodies (Goetze, et al., (2011) Glycobiology 21,949-59; Yu, et al., (2012) MAbs 4, 475-87). Therefore, methods forcontrolling the high mannose glycoform content of therapeutic proteinswould be beneficial.

There is a need in the pharmaceutical industry to manipulate and controlthe high mannose glycoform content of recombinant therapeuticglycoproteins and methods for doing such would be useful. The inventionprovides a method for manipulating the high mannose glycoform content ofrecombinant glycoproteins by regulating ornithine metabolism in the hostcells.

SUMMARY OF THE INVENTION

The invention provides a method for manipulating the high mannoseglycoform content of a recombinant protein comprising culturing a hostcell expressing the recombinant protein in a cell culture underconditions that regulate ornithine metabolism in the host cell.

In one embodiment ornithine metabolism in the host cell is regulated bydecreasing the accumulation of ornithine in the host cell. In a relatedembodiment ornithine accumulation in the host cell is regulated byculturing the host cell in a cell culture medium containing an arginaseinhibitor or spermine. In another related embodiment ornithineaccumulation in the host cell is regulated through the addition of anarginase inhibitor to cell culture medium. In another related embodimentthe arginase inhibitor is BEC (S-(2-boronoethyl)-1-cysteine) orDL-a-Difluoromethylornithine. In another related embodiment the arginaseinhibitor is BEC (S-(2-boronoethyl)-1-cysteine). In another relatedembodiment the arginase inhibitor is DL-a-Difluoromethylornithine. Inanother related embodiment the concentration of the arginase inhibitoris at least 10 μM. In another related embodiment the concentration ofthe arginase inhibitor is from 10 μM to 2 mM. In yet another relatedembodiment the concentration of the arginase inhibitor is 10 μM. In yetanother related embodiment the concentration of the arginase inhibitoris 0.5 mM. In yet another related embodiment the concentration of thearginase inhibitor is 1 mM. In yet another related embodiment theconcentration of the arginase inhibitor is 2 mM.

In another embodiment ornithine accumulation in the host cell isregulated by adding 35 μM or less of spermine to the cell culturemedium. In a related embodiment the concentration of spermine is 7 μM to35 μM. In another related embodiment the concentration of spermine is 17μM to 35 μM. In another related embodiment the concentration of spermineis 7 μM to 17 μM. In another related embodiment the concentration ofspermine is 35 μM. In another related embodiment the concentration ofspermine is 17 μM. In another related embodiment the concentration ofspermine is 7 μM.

In another embodiment ornithine metabolism in the host cell is regulatedby increasing the accumulation of ornithine in the host cell. In arelated embodiment ornithine accumulation in the host cell is regulatedby culturing the host cell in a cell culture medium containingornithine, arginine, an ornithine decarboxylase inhibitor, an ornithineaminotransferase, a nitric oxide synthase inhibitor or an argininedecarboxylase inhibitor. In a yet another related embodiment ornithineaccumulation in the host cell is regulated by the addition of at least0.6 mM ornithine to the cell culture medium. In a yet another relatedembodiment the concentration of ornithine is from 0.6 to 14.8 mM. In ayet another related embodiment the concentration of ornithine is from 6to 14.8 mM. In a yet another related embodiment the concentration ofornithine is 0.6 mM. In a yet another related embodiment theconcentration of ornithine is 6 mM.

In a yet another related embodiment the concentration of ornithine is14.8 mM. In another embodiment ornithine accumulation in the host cellis regulated by the addition of at least 8.7 mM arginine to cell culturemedium. In a yet another related embodiment the concentration ofarginine is from 8.7 mM to 17.5 mM. In a yet another related embodimentthe concentration of arginine is 8.7 mM. In a yet another relatedembodiment the concentration of arginine is 17.5 mM.

In another embodiment ornithine accumulation in the host cell isregulated through the addition of an ornithine decarboxylase inhibitor,a nitric oxide synthase inhibitor, an ornithine aminotransferaseinhibitor, or an arginine decarboxylase inhibitor to the cell culturemedium. In a related embodiment ornithine accumulation in the host cellis regulated through the addition of an ornithine decarboxylaseinhibitor to the cell culture medium. In yet another related embodimentthe ornithine decarboxylase inhibitor is alpha-defluoromethylornithine(DMFO). In yet another related embodiment the ornithine decarboxylaseinhibitor is piperonyl butoxide (PBO).

In another related embodiment ornithine accumulation in the host cell isregulated through the addition of an ornithine aminotransferaseinhibitor to the cell culture medium. In yet another related embodimentthe ornithine aminotransferase inhibitor is 5-fluoromethylornithine(F-FMOrn). In yet another related embodiment the host cell is regulatedthrough the addition of a nitric oxide synthase inhibitor to the cellculture medium. In yet another related embodiment the nitric oxidesynthase inhibitor is 2-ethyl-2-thiopseudourea or N-Nitro-L-arginine andL^(G)-monomethyl-L-arginine. In yet another related embodiment thenitric oxide synthase inhibitor is N-Nitro-L-arginine andL^(G)-monomethyl-L-arginine.

In another related embodiment ornithine accumulation in the host cell isregulated through the addition of an arginine decarboxylase inhibitor tothe cell culture medium. In yet another related embodiment the argininedecarboxylase inhibitor is asymmetric dimethyl-arginine (ADMA).

The invention provides a method of producing a recombinant proteinwherein the high mannose glycoform content is reduced comprisingculturing a host cell which expresses the recombinant protein in a cellculture wherein ornithine metabolism is regulated by reducing ornithineaccumulation in the host cell. In a related embodiment ornithineaccumulation in the host cell is reduced by culturing the host cell in acell culture medium containing an arginase inhibitor or spermine.

In a related embodiment ornithine accumulation in the host cell isreduced through the addition of an arginase inhibitor to the cellculture medium. In yet another related embodiment the arginase inhibitoris BEC (S-(2-boronoethyl)-1-cysteine) or DL-a-Difluoromethylornithine.In yet another related embodiment the arginase inhibitor is BEC(S-(2-boronoethyl)-1-cysteine). In yet another related embodiment thearginase inhibitor is DL-a-Difluoromethylornithine. In yet anotherrelated embodiment the arginase inhibitor is at least 10 μM. In yetanother related embodiment the arginase inhibitor is from 10 μM to 2 mM.In yet another related embodiment the arginase inhibitor is from 10 μMto 20 μM. In yet another related embodiment the arginase inhibitor is 10μM. In yet another related embodiment the arginase inhibitor is 0.5 mM.In yet another related embodiment the arginase inhibitor is 1 mM. In yetanother related embodiment the arginase inhibitor is 2 mM.

In another embodiment ornithine accumulation in the host cell is reducedby culturing the host cell in a cell culture medium containing 35 μM orless of spermine in the cell culture medium. In yet another relatedembodiment the concentration of spermine is 7 μM to 35 μM. In yetanother related embodiment the concentration of spermine is 17 μM to 35μM. In yet another related embodiment the concentration of spermine is0.07 mL/L to 0.17 mL/L. In yet another related embodiment theconcentration of spermine is 35 μM. In yet another related embodimentthe concentration of spermine is 17 μM. In yet another relatedembodiment the concentration of spermine is 7 μM.

The invention provides a method of producing a recombinant proteinwherein the high mannose glycoform content is increased comprisingculturing a host cell which expresses the recombinant protein in a cellculture wherein ornithine metabolism is regulated by increasingornithine accumulation in the host cell. In a related embodimentornithine accumulation in the host cell is increased by addingornithine, arginine, an ornithine decarboxylase inhibitor, an ornithineaminotransferase, a nitric oxide synthase inhibitor or an argininedecarboxylase inhibitor to the cell culture medium.

In another related embodiment ornithine accumulation in the host cell isincreased by the culturing the host cell in a cell culture mediumcontaining of at least 0.6 mM ornithine. In yet another relatedembodiment the concentration of ornithine is from 0.6 to 14.8 mM. In yetanother related embodiment the concentration of ornithine is from 6 to14.8 mM. In yet another related embodiment the concentration ofornithine is 0.6 mM. In yet another related embodiment the concentrationof ornithine is 6 mM. In yet another related embodiment theconcentration of ornithine is 14.8 mM.

In another related embodiment ornithine accumulation in the host cell isincreased by culturing the host cell in a cell culture medium containingat least 8.7 mM arginine. In yet another related embodiment theconcentration of arginine is from 8.7 mM to 17.5 mM. In yet anotherrelated embodiment the concentration of arginine is 8.7 mM. In yetanother related embodiment the concentration of arginine is 17.5 mM.

In another related embodiment ornithine accumulation is increased in thehost cell by culturing the host cell in a cell culture medium containingan ornithine decarboxylase inhibitor, a nitric oxide synthase inhibitor,an ornithine aminotransferase inhibitor, or an arginine decarboxylaseinhibitor. In another related embodiment ornithine accumulation in thehost cell-cell is increased by culturing the host cell in a cell culturemedium containing an ornithine decarboxylase inhibitor. In yet anotherrelated embodiment the ornithine decarboxylase inhibitor isalpha-defluoromethylornithine (DMFO). In yet another related embodimentthe ornithine decarboxylase inhibitor is piperonyl butoxide (PBO).

In another related embodiment ornithine accumulation in the host cell isincreased by culturing the host cell in a cell culture medium containingan ornithine aminotransferase inhibitor. In yet another relatedembodiment the ornithine aminotransferase inhibitor is5-fluoromethylornithine (F-FMOrn).

In another related embodiment ornithine accumulation in a host cell isincreased by culturing the host cell in a cell culture medium containinga nitric oxide synthase inhibitor. In yet another related embodiment thenitric oxide synthase inhibitor is 2-ethyl-2-thiopseudourea orN-Nitro-L-arginine and L^(G)-monomethyl-L-arginine.

In another embodiment ornithine accumulation in the host cell isincreased by culturing the host cell in a cell culture medium containingan arginine decarboxylase inhibitor. In yet another related embodimentthe arginine decarboxylase inhibitor is asymmetric dimethyl-arginine(ADMA).

In another embodiment the host cell expressing the recombinant proteinis cultured in a batch culture, fed-batch culture, perfusion culture, orcombinations thereof. In yet another related embodiment the culture is aperfusion culture. In yet another related embodiment perfusion comprisescontinuous perfusion. In yet another related embodiment the rate ofperfusion is constant. In yet another related embodiment the perfusionis performed at a rate of less than or equal to 1.0 working volumes perday. In yet another related embodiment the perfusion is accomplished byalternating tangential flow.

In another embodiment the host cell expressing the recombinant proteinis cultured in a bioreactor. In yet another related embodiment thebioreactor has a capacity of at least 500 L. In yet another relatedembodiment the bioreactor has a capacity of at least 500 L to 2000 L. Inyet another related embodiment the bioreactor has a capacity of at least1000 L to 2000 L. In yet another related embodiment the bioreactor isinoculated with at least 0.5×10⁶ cells/mL.

In another embodiment the host cell expressing the recombinant proteinis cultured in a serum-free cell culture medium. In another relatedembodiment the serum-free culture medium is a perfusion cell culturemedium. In yet another related embodiment the host cells are mammaliancells. In yet another related embodiment the host cells are ChineseHamster Ovary (CHO) cells.

In another embodiment the recombinant protein is a glycoprotein. Inanother embodiment the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine.

In another embodiment the methods above further comprise a step ofharvesting the recombinant protein produced by the host cell.

In another embodiment the recombinant protein produced by the host cellis purified and formulated into a pharmaceutically acceptableformulation.

In another embodiment is provides a recombinant protein produced by anyof the methods above. In a related embodiment the recombinant protein ispurified. In another embodiment the recombinant protein is formulatedinto a pharmaceutically acceptable formulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Ornithine metabolism overview. ARG, Arginase; AZ, Antizyme; AZIN,Antizyme inhibitor; PSC, Pyrroline-5-carboxylate; ASP, Aspartate; ORNT,Ornithine transporter; GATM, Glycine amidinotransferase; NOS, Nitricoxide synthase; OAT, Ornithine aminotransferase; ODC, ornithinedecarboxylase; OTC, ornithine transcarbamoylase; SMS, Spermine synthase;SRS, Spermidine synthase.

FIG. 2 Identification of ornithine as a metabolic marker associated withhigh mannose glycan levels. High mannose glycan levels (% HM) of thesecreted recombinant monoclonal antibodies from eight different celllines (Cell line A through H) detected on day 8 (D8, white bars), day 9(D9, gray bars) and day 10 (D10, black bars) of the fed-batch processevaluated under Media #1 (A) or Media #2 (B). Correlation between highmannose glycan levels and extracellular ornithine levels detected in thespent media (C). The average day 9 ornithine level and high mannoseglycan level from the eight cell lines were compared. Pearson's R-value,R=0.83.

FIG. 3 Correlation between high mannose and extracellular ornithinelevels: A) High mannose glycoform content detected when cell line H wasexposed to eight different production conditions (#1 thru #8). B)Corresponding extracellular relative ornithine levels. C) Correlationbetween % high mannose glycoform content and extracellular relativeornithine levels. Pearson's R-value, R=0.78.

FIG. 4 The relative mRNA expression levels of arginase 1 from cellpellets collected from eight different cell lines on days 3, 6, 8, 9 and10. The corresponding high mannose glycoform content is also shown.

FIG. 5 High mannose glycoform content on recombinant glycoproteinsexpressed by cells grown in cell culture medium containing sperminetetrahydrochloride at concentrations of 0, 7, 17, 35 and 100 μM. Sampleswere collected on day 5 of the mock perfusion assay. The 35 μM sampleserved as a control.

FIG. 6 Concentration of extracellular ornithine (mg/L) endogenouslyproduced by cell cultures exposed to spermine tetrahydrochloride atconcentrations of 0, 7, 17, 35 and 100 μM. Samples were collected on day5 of the mock perfusion assay. The 35 μM sample served as a control.

FIG. 7 High mannose glycoform content on recombinant glycoproteinsexpressed by cells grown in cell culture medium containing L-ornithinemonohydrochloride at concentrations of 0, 0.6, 6 and 14.8 mM. Sampleswere collected on day 5 of the mock perfusion assay. The 0 mM sampleserved as a control.

FIG. 8 High mannose glycoform content on recombinant glycoproteinsexpressed by Cell line “I” grown in cell culture medium containing 0.1g/L L-ornithine monohydrochloride (black bars) or control cell culturecontaining no exogenously added ornithine (white bars). Samples werecollected on days 6, 8 and 12.

FIG. 9 High mannose glycoform content on recombinant glycoproteinsexpressed by cells grown in cell culture medium containing argininemonohydrochloride at concentrations of 2.2, 4.4, 6.5, 8.7 and 17.5 mM.The 8.7 mM sample acted as control. Samples were collected on day 4 ofthe mock perfusion assay.

FIG. 10 High mannose glycoform content on recombinant glycoproteinsexpressed by cells grown in cell culture medium supplemented withvarious arginase inhibitors: BEC Hydrochloride (BEC), DL-α,Diflouromethylornithine Hydrochloride (DL-a), N^(G)-Hydroxy-L-arginineMonoacetate salt (NG) and Nω-Hydroxy-nor-arginine diacetate salt (Nw) atconcentrations of 1, 10 and 20 μM. Control contained no inhibitor.Samples were collected on day 4 of the mock perfusion assay.

FIG. 11 High mannose glycoform content on recombinant glycoproteinsexpressed by cells grown in cell culture medium containing the arginaseinhibitor BEC Hydrochloride (BEC) at concentrations of 0.0 0.01 and 0.5mM and, DL-α, Diflouromethylornithine Hydrochloride (DL-a) atconcentrations of 0.0, 0.01, 1.0 and 2.0 mM. Samples were collected onday 4 of the mock perfusion assay.

DETAILED DESCRIPTION OF THE INVENTION

It was found that the high mannose glycoform content of an expressedrecombinant glycoprotein was influenced by ornithine accumulation in thehost cell and as a result could be manipulated by regulating ornithinemetabolism in the host cell.

Ornithine is a non-protein coding amino acid involved in the urea cycle,polyamine synthesis and arginine metabolism. Ornithine is also aprecursor of glutamate and proline via ornithine-δ-aminotransferase(OAT) activities, see FIG. 1. In humans, deficiency of OAT results ingyrate atrophy of the choroid and retina (GA), a disorder characterizedby retinal degeneration and plasma ornithine accumulation (Takki K etal., Br J Ophthalmol. 1974; 58(11): 907-16). In a mouse model ofOAT-deficiency, arginine restricted diet has shown to reduce plasmaornithine levels and prevent retinal degeneration (Wang T et al., PNAS2000; 97(3): 1224-1229). Ornithine decarboxylase (ODC), which catalyzesthe conversion of ornithine to putrescine, is the rate limiting enzymeof the polyamine biosynthetic pathway (Pegg A, JBC. 2006; 281(21):14529-14532). ODC synthesis and stability, as well as polyaminetransporter activity, is affected by environmental osmotic conditions(Munro G et al., BBA 1975; 411(2): 263-281; Tohyama et al., Eur JBiochem. 1991; 202(3):1327-1331; Michell J et al., 1998; 329:453-459).Increased polyamine biosynthesis has been associated with increasedresistance to osmotic stress in plants (Alcazar R et al., BiotechnolLett 2006; 28:1867-1876). Conversion of ornithine to citrulline iscatalyzed by ornithine transcarbamylase (OTC) as part of urea cycle. OTCdeficiency in humans causes accumulation of ammonia in the blood(Hopkins et al., Arch. Dis. Childh., 1969 44:143-148). Ornithinemetabolism occurs in both cytosol and mitochondria, with OTC and OATcatalyzed metabolic steps taking place in the mitochondria.Mitochondrial ornithine transporter ORNT1 is required for the import ofornithine into the mitochondria. In humans, mutations in ORNT1 areresponsible for hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)syndrome which is characterized by elevated plasma levels of ornithineand ammonia (Camacho et al., Nat Genet (1999); 22:151-158); (Valle D etal, 2001, 1857-1896).

As described herein, the degree of ornithine accumulation in the hostcell, as measured by extracellular levels of ornithine in cell culturemedium, was found to correlate with high mannose glycoform content ofexpressed recombinant glycoproteins. Manipulating the high mannosecontent could be accomplished by regulating ornithine metabolism in thehost cell. The invention provides a method for manipulating the highmannose glycoform content of a recombinant protein comprising culturinga host cell expressing the recombinant protein in a cell culture underconditions that regulate ornithine metabolism in the host cell.Ornithine metabolism can be regulated by decreasing or increasingaccumulation of ornithine in the host cell. Methods are provided hereinfor producing recombinant proteins wherein the high mannose glycoformcontent is reduced or increased comprising culturing a host cell whichexpresses the recombinant protein in a cell culture wherein ornithineaccumulation in the host cell is regulated.

Ornithine metabolism refers to chemical or enzymatic reactions andpathways involved in the ornithine biosynthesis, transport, catabolicprocess and metabolic conversions. The urea cycle, polyamine synthesis,creatine synthesis, and the mitochondrial ornithine catabolism pathwaysare the examples of ornithine metabolism. An overview is provided inFIG. 1.

Ornithine accumulation in a host cell is the consequence of alteredornithine metabolism. The extent of ornithine accumulation in the hostcell can be modulated by regulating ornithine metabolism. Intracellularmetabolite levels can be reflected in extracellular levels (i.e.,detected in the cell culture medium). An indicator of the accumulationof ornithine accumulation in a host cell can be made by measuring theamount of ornithine secreted into the cell culture medium. As describedherein, time-course dependent increases in ornithine levels were foundin cell culture media lacking exogenous ornithine.

“High mannose glycoform content”, “high mannose glycan level” and“levels of high mannose species” are used interchangeably and designatedby the abbreviations “HM”, “% HM”, “HMN” or “% HMN” and refer to therelative percentage of mannose 5 (Man5), mannose 6 (Man6), mannose 7(Man7), mannose 8 (Man8) and mannose 9 (Man9) glycan species combined.

It was found that the level of ornithine secreted into cell culturemedium correlated with high mannose glycoform content of the recombinantglycoproteins expressed by host cells in the cell culture. When theaccumulation of ornithine in a host cell was decreased by culturing thehost cell in a cell culture medium containing an arginase inhibitor orspermine, the high mannose glycoform content of the expressedglycoproteins was decreased. When ornithine accumulation in the hostcell was increased by culturing the host cell in a cell culture mediumcontaining ornithine or arginine, the high mannose glycoform content ofthe expressed glycoproteins was increased.

The invention provides a method for regulating ornithine accumulation ina host cell by culturing the host cell in a cell culture mediumcontaining an arginase inhibitor. Arginine is the metabolic precursor ofornithine and arginase is an enzyme which catalyzes the conversion ofarginine into ornithine. It was observed that arginase mRNA expressionlevels correlated with the amount of ornithine accumulation when themetabolic and expression profiles of different cell lines were compared.Blocking the activity of arginase with an arginase inhibitor couldpotentially reduce ornithine production levels. However, theeffectiveness of arginase inhibitors may be compromised due to the highlevel of arginine in cell culture medium. In addition, there are othermetabolic precursors of ornithine (i.e. glutamate and proline, seeFIG. 1) that may contribute to ornithine accumulation.

As described herein, it was found that high mannose glycoform content ofa recombinant protein expressed by the cultured host cell could bemodulated by adding an arginase inhibitor to the cell culture medium.Blocking the activity of arginase reduced the amount of ornithineproduction in the host cells, lowered the high mannose glycan level ofthe expressed recombinant glycoproteins.

Arginase also inhibits ornithine transcarbamoylase (OTC) (Vissers etal., (1982) J. Gen. Microbio. 128:1235-1247). Blocking the activity ofarginase not only reduces ornithine production from arginine but couldpotentially relieve the repression of OTC activity, allowing ornithineconversion to citrulline, allowing for additional reduction of ornithineaccumulation (see FIG. 1).

In patients with OTC deficiency, ammonia levels are increased. Ifenhanced arginase expression or activity in host cell lines expressingrecombinant proteins with high levels of high mannose glycans inducesOTC inhibition, this m also result in ammonia level increase. Increasein the intracellular levels of ammonia could potentially alter pHgradient in Golgi apparatus and induce suboptimal relocalization ofglycosyltransferases, resulting in higher levels of high mannose glycansdue to incomplete glycan branching in Golgi complex (Campbell et al,(1973) NJM 288 (1):1-6; Hopkins et al., (1969) Archive of Disease inChildhood 44(234):143-148; Willing et al, (2001) Amino Acids21(3):303-318; Park et al., (2000) J. Biotechnol 81(2):129-140; Rivinojaet al., (2009) J. Cell Physiol. 220(1):144-154; and Axelsson et al.,(2001) Glycobiology 11(8): 633-644).

Useful arginase inhibitors are known in the art and available fromcommercial sources. Such arginase inhibitors include, BEC Hydrochloride;DL-α, Diflouromethylornithine Hydrochloride; N^(G)-Hydroxy-L-arginineand Nω-Hydroxy-nor-arginine. In one embodiment the arginase inhibitor isBEC (S-(2-boronoethyl)-1-cysteine) or DL-a-Difluoromethylornithine. Inone embodiment of the invention the arginase inhibitor is BEC(S-(2-boronoethyl)-1-cysteine). In one embodiment of the invention thearginase inhibitor is DL-a-Difluoromethylornithine.

Arginase inhibitors can be added to cell culture medium atconcentrations of at least 10 μM to decrease high mannose glycan levelsof expressed recombinant proteins without significantly affectingproductivity. In one embodiment, the concentration of the arginaseinhibitor is from 10 μM to 2 mM. In another embodiment, theconcentration of the arginase inhibitor is 10 μM. In another embodimentthe concentration of the arginase inhibitor is 0.5 mM. In anotherembodiment the concentration of the arginase inhibitor is 1 mM. Inanother embodiment the concentration of the arginase inhibitor is 2 mM.

Ornithine accumulation in the host cell can also be regulated byculturing the host cell in a cell culture medium containing spermine, asdescribed in the Examples below. Ornithine, via the action of ornithinedecarboxylase (ODC), is the starting point for the polyamine pathway andsynthesis of the polyamines putrescine, spermidine and spermine.Exogenously added spermine can be used to inactivate ODC directly, orthrough antizyme, see FIG. 1. Inactivation of ODC can lead toaccumulation of ornithine as described below. By limiting the amount ofexogenously added spermine, suppression of ODC activity can be relievedand ornithine can be metabolized through the polyamine pathway, therebyreducing the overall ornithine accumulation in the host cell.

Spermine can be added to cell culture medium at concentrations of lessthan or equal to 35 μM to decrease high mannose glycan levels ofexpressed recombinant proteins without significantly affectingproductivity. In one embodiment the concentration of spermine is 7 μM to35 μM. In one embodiment the concentration of spermine is 17 μM to 35μM. In one embodiment the concentration of spermine is 7 μM to 17 μM. Inanother embodiment the concentration of spermine is 35 μM. In anotherembodiment the concentration of spermine is 17 μM. In another embodimentthe concentration of spermine is 7 μM.

Another method for regulating ornithine metabolism is to increase theaccumulation of ornithine. In one embodiment the invention providesregulating ornithine accumulation in a host cell by culturing the hostcell in a cell culture medium containing ornithine, arginine, anornithine decarboxylase inhibitor, an ornithine aminotransferase, anitric oxide synthase inhibitor or an arginine decarboxylase inhibitor.

As described herein, levels of extracellular ornithine in conditionedcell culture media was found to correlate to high mannose glycan levelson recombinant glycoproteins. It was found that culturing host cellsexpressing recombinant proteins in a cell culture medium containingornithine resulted in recombinant glycoproteins having increased levelsof high mannose glycans.

As described above, ornithine accumulation in cell culture media likelyreflects alterations in ornithine metabolism which can lead toaccumulation of ammonia, similar to patients carrying defective genes ofornithine metabolism (eg. OTC deficiency or ORNT1 mutation). While thecellular mechanism behind ammonia induced high mannose increase is notknown, it has been suggested that alteration in the pH gradient in Golgican lead to suboptimal relocalization of glycosyltransferases. Thesechanges could lead to decreased availability of glycosylation enzymes tocomplete glycan branching, and thus result in higher levels of highmannose glycan levels.

Another possibility is that ornithine accumulation potentially inducesdisturbance of redox homeostasis (Zanatta et al., (2013) Life sciences93(4): 161-168). Ornithine correlates positively with high mannosesuggesting the possibility that high mannose glycoform content isregulated by cellular redox state. Ornithine can increase the levels oflipid oxidation. Since many of the glycosylation regulating enzymes arelipid membrane bound, alterations in lipid oxidation caused by ornithineaccumulation could potentially alter the integrity and activity ofglycosylation regulating enzymes in Golgi and ER, and subsequentlyaffect high mannose glycoform contents.

Ornithine can be added to cell culture medium at concentrations of atleast 0.6 mM, to increase high mannose glycan levels of expressedrecombinant proteins without significantly affecting productivity. Inone embodiment the concentration of ornithine is from 0.6 mM to 14.8 mM.In one embodiment the concentration of ornithine is from 6 mM to 14.8mM. In another embodiment the concentration of ornithine is 0.6 mM. Inanother embodiment the concentration of ornithine is 6 mM. In anotherembodiment the concentration of ornithine is 14.8 mM.

Arginine is the metabolic precursor of ornithine. It was found that highmannose glycan levels were increased in recombinant proteins expressedin host cells cultivated in cell culture medium containing exogenousarginine. Increasing the amount of exogenous arginine increased theamount of metabolic precursor available for ornithine synthesis, therebyincreasing the levels of ornithine in host cells.

The invention provides regulating ornithine accumulation in host cellsby adding arginine at concentrations of at least 8.7 mM to increase highmannose glycan levels of expressed recombinant proteins withoutsignificantly affecting productivity. In one embodiment theconcentration of arginine is from 8.7 to 17.5 mM. In another embodimentthe concentration of arginine is 8.7 mM. In another embodiment theconcentration of arginine is 17.5 mM.

Ornithine accumulation may be regulated through the addition to cellculture medium of inhibitors of Ornithine decarboxylase (ODC), Ornithineaminotransferase (OAT), Nitric oxide synthase (NOS) or ArginineDecarboxylase (ADC), thereby providing a way to regulate ornithinemetabolism and accumulation of ornithine in the host cell to manipulatethe high mannose glycoform content of a recombinant protein.

Ornithine accumulation can be increased by blocking the activity ofornithine metabolizing enzymes such as ODC and OAT (see FIG. 1).Small-molecule inhibitors specific for ODC such asalpha-difluoromethylornithine (DFMO) and piperonyl butoxide (PBO) arecommercially available. OAT is blocked by 5-fluoromethylornithine(5-FMOrn) T (Daune et al., 1988, Biochem J. 253:481-488). The inventionprovides supplementing a cell culture with these inhibitors to increaseornithine accumulation in the host cells to manipulate the high mannoseglycoform content of a recombinant protein.

Ornithine accumulation may be regulated by blocking the activity of theenzymes that regulate arginine accumulation (FIG. 1). Inhibiting theactivity of nitric oxide synthase by small molecule inhibitors such as2-ethyl-2-thiopseudourea and N-Nitro-L-arginine andL^(G)-monomethyl-L-arginine and/or arginine decarboxylase activity byasymmetric dimethyl-arginine (ADMA) may enhance flux of ornithineconversion from arginine. The invention provides supplementing a cellculture with these inhibitors to increase in ornithine accumulation inthe host cell to manipulate the high mannose glycoform content of arecombinant protein.

In one embodiment of the invention is provided that the cell culturemedium is a serum-free cell culture medium. In one embodiment the cellculture medium is a perfusion cell culture medium.

As used herein, the terms “cell culturing medium” (also called “culturemedium,” “cell culture media,” “tissue culture media,”) refers to anynutrient solution used for growing cells, e.g., animal or mammaliancells, and which generally provides at least one or more components fromthe following: an energy source (usually in the form of a carbohydratesuch as glucose); one or more of all essential amino acids, andgenerally the twenty basic amino acids, plus cysteine; vitamins and/orother organic compounds typically required at low concentrations; lipidsor free fatty acids; and trace elements, e.g., inorganic compounds ornaturally occurring elements that are typically required at very lowconcentrations, usually in the micromolar range.

The nutrient solution may optionally be supplemented with additionalcomponents to optimize growth of cells, such as hormones and othergrowth factors, e.g., insulin, transferrin, epidermal growth factor,serum, and the like; salts, e.g., calcium, magnesium and phosphate, andbuffers, e.g., HEPES; nucleosides and bases, e.g., adenosine, thymidine,hypoxanthine; and protein and tissue hydrolysates, e.g., hydrolyzedanimal protein (peptone or peptone mixtures, which can be obtained fromanimal byproducts, purified gelatin or plant material); antibiotics,e.g., gentamycin; ell protectants or surfactants, e.g., Pluronic® F68;polyamines, e.g., putrescine, spermidine or spermine (see e.g., WIPOPublication No. WO 2008/154014) and pyruvate (see e.g. U.S. Pat. No.8,053,238) depending on the requirements of the cells to be culturedand/or the desired cell culture parameters.

Cell culture medium include those that are typically employed in and/orare known for use with any cell culture process, such as, but notlimited to, batch, extended batch, fed-batch and/or perfusion orcontinuous culturing of cells.

Cell culture medium components may be completely milled into a powdermedium formulation; partially milled with liquid supplements added tothe cell culture medium as needed; or nutrients may be added in acompletely liquid form to the cell culture.

A “base” (or batch) cell culture medium refers to a cell culture mediumthat is typically used to initiate a cell culture and is sufficientlycomplete to support the cell culture.

A “growth” cell culture medium refers to a cell culture medium that istypically used in cell cultures during a period of exponential growth, a“growth phase”, and is sufficiently complete to support the cell cultureduring this phase. A growth cell culture medium may also containselection agents that confer resistance or survival to selectablemarkers incorporated into the host cell line. Such selection agentsinclude, but are not limited to, geneticin (G4118), neomycin, hygromycinB, puromycin, zeocin, methionine sulfoximine, methotrexate,glutamine-free cell culture medium, cell culture medium lacking glycine,hypoxanthine and thymidine, or thymidine alone.

A “production” cell culture medium refers to a cell culture medium thatis typically used in cell cultures during the transition and productionphases when exponential growth is ending and protein production takesover, and is sufficiently complete to maintain a desired cell density,viability and/or product titer these phases.

A “perfusion” cell culture medium refers to a cell culture medium thatis typically used in cell cultures that are maintained by perfusion orcontinuous culture methods and is sufficiently complete to support thecell culture during this process. Perfusion cell culture mediumformulations may be enriched or more concentrated than base cell culturemedium formulations to accommodate for the method used to remove thespent medium. Perfusion cell culture medium can be used during both thegrowth and production phases.

Concentrated cell culture medium can contain some or all of thenutrients necessary to maintain the cell culture; in particular,concentrated medium can contain nutrients identified as or known to beconsumed during the course of the production phase of the cell culture.Concentrated medium may be based on just about any cell culture mediaformulation. Such a concentrated feed medium can contain some or all thecomponents of the cell culture medium at, for example, about 2×, 3×, 4×,5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×,600×, 800×, or even about 1000× of their normal amount.

Cell cultures can also be supplemented with independent concentratedfeeds of particular nutrients which may be difficult to formulate or arequickly depleted in cell cultures. Such nutrients may be amino acidssuch as tyrosine, cysteine and/or cystine (see e.g., WIPO PublicationNo. 2012/145682). In one embodiment, a concentrated solution of tyrosineis independently fed to a cell culture grown in a cell culture mediumcontaining tyrosine, such that the concentration of tyrosine in the cellculture does not exceed 8 mM. In another embodiment, a concentratedsolution of tyrosine and cystine is independently fed to the cellculture being grown in a cell culture medium lacking tyrosine, cystineor cysteine. The independent feeds can begin prior to or at the start ofthe production phase. The independent feeds can be accomplished by fedbatch to the cell culture medium on the same or different days as theconcentrated feed medium. The independent feeds can also be perfused onthe same or different days as the perfused medium.

Cell culture medium, in certain embodiments, is serum-free and/or freeof products or ingredients of animal origin. Cell culture medium, incertain embodiments, is chemically defined, where all of the chemicalcomponents are known.

As is appreciated by the practitioner, animal or mammalian cells arecultured in a medium suitable for the particular cells being culturedand which can be determined by the person of skill in the art withoutundue experimentation. Commercially available media can be utilized andinclude, but is not limited to, Iscove's Modified Dulbecco's Medium,RPMI 1640, Minimal Essential Medium-alpha. (MEM-alpha), Dulbecco'sModification of Eagle's Medium (DMEM), DME/F12, alpha MEM, Basal MediumEagle with Earle's BSS, DMEM high Glucose, with Glutamine, DMEM highglucose, without Glutamine, DMEM low Glucose, without Glutamine,DMEM:F12 1:1, with Glutamine, GMEM (Glasgow's MEM), GMEM with glutamine,Grace's Complete Insect Medium, Grace's Insect Medium, without FBS,Ham's F-10, with Glutamine, Ham's F-12, with Glutamine, IMDM with HEPESand Glutamine, IMDM with HEPES and without Glutamine, IP41 InsectMedium, 15 (Leibovitz)(2×), without Glutamine or Phenol Red, 15(Leibovitz), without Glutamine, McCoy's 5A Modified Medium, Medium 199,MEM Eagle, without Glutamine or Phenol Red (2×), MEM Eagle-Earle's BSS,with glutamine, MEM Eagle-Earle's BSS, without Glutamine, MEMEagle-Hanks BSS, without Glutamine, NCTC-109, with Glutamine, Richter'sCM Medium, with Glutamine, RPMI 1640 with HEPES, Glutamine and/orPenicillin-Streptomycin, RPMI 1640, with Glutamine, RPMI 1640, withoutGlutamine, Schneider's Insect Medium or any other media known to oneskilled in the art, which are formulated for particular cell types. Tothe foregoing exemplary media can be added supplementary components oringredients, including optional components, in appropriateconcentrations or amounts, as necessary or desired, and as would beknown and practiced by those having in the art using routine skill.

In one embodiment of the invention is provided that the host cells aremammalian cells. In one embodiment the host cells are Chinese HamsterOvary (CHO) cells.

Cell lines (also referred to as “host cells”) used in the invention aregenetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. The cells can contain introduced, e.g., viatransformation, transfection, infection, or injection, expressionvectors (constructs), such as plasmids and the like, that harbor codingsequences, or portions thereof, encoding the proteins for expression andproduction in the culturing process. Such expression vectors contain thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to and practiced by thoseskilled in the art can be used to construct expression vectorscontaining sequences encoding the produced proteins and polypeptides, aswell as the appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in J. Sambrook et al., 2012, Molecular Cloning, ALaboratory Manual, 4^(th) edition Cold Spring Harbor Press, Plainview,N.Y. or any of the previous editions; F. M. Ausubel et al., 2013,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., or any of the previous editions; Kaufman, R. J., Large ScaleMammalian Cell Culture, 1990, all of which are incorporated herein forany purpose.

Animal cells, mammalian cells, cultured cells, animal or mammalian hostcells, host cells, recombinant cells, recombinant host cells, and thelike, are all terms for the cells that can be cultured according to theprocesses of this invention. Such cells are typically cell linesobtained or derived from mammals and are able to grow and survive whenplaced in either monolayer culture or suspension culture in mediumcontaining appropriate nutrients and/or other factors, such as thosedescribed herein. The cells are typically selected that can express andsecrete proteins, or that can be molecularly engineered to express andsecrete, large quantities of a particular protein, more particularly, aglycoprotein of interest, into the culture medium. It will be understoodthat the protein produced by a host cell can be endogenous or homologousto the host cell. Alternatively, the protein is heterologous, i.e.,foreign, to the host cell, for example, a human protein produced andsecreted by a Chinese hamster ovary (CHO) host cell. Additionally,mammalian proteins, i.e., those originally obtained or derived from amammalian organism, are attained by the methods the present inventionand can be secreted by the cells into the culture medium.

The methods of the present invention can be used in the culture of avariety of cells. In one embodiment, the cultured cells are eukaryoticcells such as plant and/or animal cells. The cells can be mammaliancells, fish cells, insect cells, amphibian cells or avian cells. A widevariety of mammalian cell lines suitable for growth in culture areavailable from the American Type Culture Collection (Manassas, Va.) andother depositories as well as commercial vendors. Cell that can be usedin the processes of the invention include, but not limited to, MK2.7cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1(ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet.,12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/−DHFR,Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), anddp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCCCCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7,ATCC CRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8 hybridoma cells,Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkatcells, THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells) and established cell lines and their strains (e.g.,human embryonic kidney cells (e.g., 293 cells, or 293 cells subclonedfor growth in suspension culture, Graham et al., 1977, J. Gen. Virol.,36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse sertolicells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervicalcarcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCCCCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells(HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51);buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather,1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells,BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells,WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells, 1-10 cells,RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15) cells, GELcells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof),fibroblast cells from any tissue or organ (including but not limited toheart, liver, kidney, colon, intestines, esophagus, stomach, neuraltissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells,Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells,Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells,WI-38 cells, WI-26 cells, MiCl₁ cells, CV-1 cells, COS-1 cells, COS-3cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCCCRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C₃H/IOTI/2 cells, HSDM₁C₃ cells, KLN205 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells,Swiss/3T3 cells, Indian muntac cells, SIRC cells, C_(II) cells, andJensen cells, or derivatives thereof) or any other cell type known toone skilled in the art.

Cells may be suitable for adherent, monolayer or suspension culture,transfection, and expression of proteins, for example, antibodies. Thecells can be used with batch, fed batch and perfusion or continuousculture methods.

In one embodiment of the invention the host cell expressing therecombinant protein is cultured in a bioreactor. In another embodimentof the invention the bioreactor has a capacity of at least 500 L. In arelated embodiment the bioreactor has a capacity of at least 500 L to2000 L. In yet another related embodiment the bioreactor has a capacityof at least 1000 L to 2000 L. In one embodiment of the invention thecell culture is established by inoculating the bioreactor with at least0.5×10⁶ cells/mL in a serum-free culture medium. In one embodiment, theinvention further comprising a step of harvesting the recombinantprotein produced by the host cell. In one embodiment the inventionprovides that the recombinant protein produced by the host cell ispurified and formulated into a pharmaceutically acceptable formulation.

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,batch culture, extended batch, fed-batch culture, perfusion culture, orcombinations thereof. In batch culture, cells are initially cultured inmedium and this medium is not removed, replaced, or supplemented, i.e.,the cells are not “fed” with fresh medium, during or before the end ofthe culturing run. The desired product is harvested at the end of theculturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run, i.e., the cells are“fed” with new medium (“fed medium”) during the culturing period.Fed-batch cultures can include the various feeding regimens and times asdescribed above, for example, daily, every other day, every two days,etc., more than once per day, or less than once per day, and so on.Further, fed-batch cultures can be fed continuously with feeding medium.The desired product is then harvested at the end of theculturing/production run.

Perfusion culture is one in which the cell culture receives freshperfusion medium and spent medium is removed. Perfusion of fresh mediainto the cell culture and removal of spend media can be continuous,step-wise, intermittent, or a combination of any or all of these.Perfusion rates can range from less than one working volume per day tomany working volumes per day. Preferably the cells are retained in theculture and the spent medium that is removed is substantially free ofcells or has significantly fewer cells than the culture. Recombinantproteins expressed by the cell culture can also be retained in theculture or removed with the spent medium. Removal of the spent mediummay be accomplished by a number of means including centrifugation,sedimentation, or filtration, See e.g. Voisard et al., (2003),Biotechnology and Bioengineering 82:751-65. A preferred filtrationmethod is alternating tangential flow filtration. The alternatingtangential flow is maintained by pumping medium through hollow-fiberfilter modules using an ATF device. See e.g. U.S. Pat. No. 6,544,424;Furey (2002) Gen. Eng. News. 22 (7), 62-63. The filters separateparticles on basis of size or molecular weight. Depending on theapplication, filters may be chosen based on pore size or a molecularweight cut off (MWCO) value. Filters include membrane filters, ceramicfilters and metal filters and may be in any shape, including spiralwound or tubular or in the form of a sheet.

The term “perfusion flow rate” is the amount of media that is passedthrough (added and removed) from a bioreactor, typically expressed assome portion of or a multiple of the working volume, in a given time.“Working volume” refers to the amount of bioreactor volume used for cellculture. In one embodiment the perfusion flow rate is one working volumeor less per day.

Cell culture can be carried out under conditions for small to largescale production of recombinant proteins using culture vessels and/orculture apparatuses that are conventionally employed for animal ormammalian cell culture. As is appreciated by those having skill in theart, tissue culture dishes, T-flasks and spinner flasks are typicallyused on a laboratory bench scale. For culturing on a larger scaleequipment such as. but not limited to, fermentor type tank culturedevices, air lift type culture devices, fluidized bed bioreactors,hollow fiber bioreactors, roller bottle cultures, stirred tankbioreactor systems, packed bed type culture devices, and single usedisposable bags or any other suitable devise known to one skilled in theart can be used. Microcarriers may or may not be used with the rollerbottle or stirred tank bioreactor systems. The systems can be operatedin a batch, fed-batch or perfusion/continuous mode. In addition, theculture apparatus or system may be equipped with additional apparatus,such a cell separators using filters, gravity, centrifugal force, andthe like.

The production of recombinant proteins can be done in multiple phaseculture processes. In a multiple phase process, cells are cultured intwo or more distinct phases. For example cells may be cultured first inone or more growth phases, under environmental conditions that maximizecell proliferation and viability, then transitioned to a productionphase, under conditions that maximize protein production. In acommercial process for production of recombinant proteins by mammaliancells, there are commonly multiple, for example, at least about 2, 3, 4,5, 6, 7, 8, 9, 10 or more growth phases that occur in different culturevessels (N-x to N-1) preceding a final production culture. The growthand production phases may be preceded by, or separated by, one or moretransition phases. A production phase can be conducted at large scale.

The term “growth phase” of a cell culture refers to the period ofexponential cell growth (i.e., the log phase) where the cells aregenerally rapidly dividing. Cells are maintained at the growth phase fora period of about one day, or about two days, or about three days, orabout four days, or longer than four days. The duration of time forwhich the cells are maintained at growth phase will vary based on thecell-type and rate of growth of cells and the culture conditions, forexample.

The term “transition phase” refers to a period of time between thegrowth phase and the production phase. Generally, transition phase isthe time during which culture conditions may be controlled to support ashift from growth phase to production phase. Various cell cultureparameters which may be controlled include but are not limited to, oneor more of, temperature, osmolality, vitamins, amino acids, sugars,peptones, ammonium and salts.

The term “production phase” of a cell culture refers to the period oftime where the cell growth has plateaued. The logarithmic cell growthtypically ends before or during this phase and protein production takesover. Fed batch and perfusion cell culture processes supplement the cellculture medium or provide fresh medium so as to achieve and maintaindesired cell density, viability and product titer at this stage. Aproduction phase can be conducted at large scale. Large scale cellcultures can be maintained in a volume of at least about 100, 500, 1000,2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In apreferred embodiment the production phase is conducted in 500 L, 1000 Land/or 2000 L bioreactors.

Typically the cell cultures that precede a final production culture gothrough two prior phases, seed and inoculum trains. The seed train phase(N-X) takes place at small scale where cells are quickly expanded innumber. At the inoculums train phase (N-1), cells are further expandedto generate the inoculum for the production bioreactor, such as aninoculums of at least 0.5×10⁶ cells/mL. Seed and N-1 trains can beproduced by any culture method, typically batch cell cultures. N-1 celldensities of >15×10⁶ cells/mL are typical for seeding productionbioreactors. Higher N-1 cell densities can decrease or even eliminatethe time needed to reach a desired cell density in the productionbioreactor. A preferred method for achieving higher N-1 cell densitiesis perfusion culture using alternating tangential flow filtration. AnN-1 cell culture grown by means of a perfusion process using alternatingtangential flow filtration can provide cells at any desired density,such as densities of >90×10⁶ cells/mL or more. The N-1 cell culture canbe used to generate a single bolus inoculation culture or can be used asa rolling seed stock culture that is maintained to inoculate multipleproduction bioreactors. The inoculation density can have a positiveimpact on the level of recombinant protein produced. Product levels tendto increase with increasing inoculation density. Improvement in titer istied not only to higher inoculation density, but is likely to beinfluenced by the metabolic and cell cycle state of the cells that areplaced into production.

The term “cell density” refers to the number of cells in a given volumeof culture medium. “Viable cell density” refers to the number of livecells in a given volume of culture medium, as determined by standardviability assays (such as trypan blue dye exclusion method). The term“packed cell volume” (PCV), also referred to as “percent packed cellvolume” (% PCV), is the ratio of the volume occupied by the cells, tothe total volume of cell culture, expressed as a percentage (seeStettler, et al., (2006) Biotechnol Bioeng. December 20: 95(6):1228-33).Packed cell volume is a function of cell density and cell diameter;increases in packed cell volume could arise from increases in eithercell density or cell diameter or both. Packed cell volume is a measureof the solid level in the cell culture.

During production, a growth phase may occur at a higher temperature thana production phase. For example, a growth phase may occur at a firsttemperature set-point from about 35° C. to about 38° C., and aproduction phase may occur at a second temperature set-point from about29° C. to about 37° C., optionally from about 30° C. to about 36° C. orfrom about 30° C. to about 34° C.

In addition, chemical inducers of protein production, such as caffeine,butyrate, and/or hexamethylene bisacetamide (HMBA), may be added at thesame time as, before, or after a temperature shift. If inducers areadded after a temperature shift, they can be added from one hour to fivedays after the temperature shift, optionally from one to two days afterthe temperature shift. The cell cultures can be maintained for days oreven weeks while the cells produce the desired protein(s).

Another method to maintain cells at a desired physiological state is toinduce cell growth-arrest by exposure of the cell culture to lowL-asparagine conditions (see e.g., WIPO Publication No. WO2013/006479).Cell growth-arrest may be achieved and maintained through a culturemedium that contains a limiting concentration of L-asparagine andmaintaining a low concentration of L-asparagine in the cell culture.Maintaining the concentration of L-asparagine at 5 mM or less can beused to maintain cells in a growth-arrested state whereby productivityincreased.

Cell cycle inhibitors, compound known or suspected to regulate cellcycle progression and the associated processes of transcription, DNArepair, differentiation, senescence and apoptosis related to this arealso useful to induce cell growth-arrest. Cell cycle inhibitors thatinteract with the cycle machinery, such as cyclin-dependent kinases(CDKs) are useful as are those molecules that interact with proteinsfrom other pathways, such as AKT, mTOR, and other pathways that affect,directly or indirectly, the cell cycle.

Cell culture conditions suitable for the methods of the presentinvention are those that are typically employed and known for batch,fed-batch, or perfusion (continuous) culturing of cells or anycombination of those methods, with attention paid to pH, dissolvedoxygen (O₂), and carbon dioxide (CO₂), agitation and humidity, andtemperature.

The methods of the invention can be used to culture cells that expressrecombinant proteins of interest. The expressed recombinant proteins maybe secreted into the culture medium from which they can be recoveredand/or collected. In addition, the proteins can be purified, orpartially purified, from such culture or component (e.g., from culturemedium) using known processes and products known in the art and/oravailable from commercial vendors. The purified proteins can then be“formulated”, meaning buffer exchanged into a pharmaceuticallyacceptable formulation, sterilized, bulk-packaged, and/or packaged for afinal user. Pharmaceutically acceptable formulations can includediluents, carriers, solubilizers, emulsifiers, preservatives, and/oradjuvants. Preparing pharmaceutically acceptable formulations is withinthe skill of one in the art and includes those described in Remington'sPharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton,Pa.

In one embodiment of the invention is provided that the recombinantprotein is a glycoprotein. In one embodiment of the invention isprovided that the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine. Also provided isa recombinant protein produced by the method of the invention. In oneembodiment the recombinant protein according is formulated into apharmaceutically acceptable formulation.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation resulting in glycoproteins,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation.

As used herein, the term “glycoprotein” refers to peptides and proteins,including antibodies, having at least one oligosaccharide side chainincluding mannose residues. Glycoproteins may be homologous to the hostcell, or may be heterologous, i.e., foreign, to the host cell beingutilized, such as, for example, a human glycoprotein produced by aChinese hamster ovary (CHO) host cell. Such glycoproteins are generallyreferred to as “recombinant glycoproteins.” In certain embodiments,glycoproteins expressed by a host cell are directly secreted into themedium.

Proteins can be of scientific or commercial interest, includingprotein-based drugs. Proteins include, among other things, antibodies,fusion proteins, and cytokines. Peptides, polypeptides and proteins maybe produced by recombinant animal cell lines using cell culture methodsand may be referred to as “recombinant peptide”, “recombinantpolypeptide” and “recombinant protein”. The expressed protein(s) may beproduced intracellularly or secreted into the culture medium from whichit can be recovered and/or collected.

Nonlimiting examples of mammalian proteins that can be advantageouslyproduced by the methods of this invention include proteins comprisingamino acid sequences identical to or substantially similar to all orpart of one of the following proteins: tumor necrosis factor (TNF), flt3ligand (WO 94/28391), erythropoietin, thrombopoietin, calcitonin, IL-2,angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322): 55-60),ligand for receptor activator of NF-kappa B (RANKL, WO 01/36637), tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO97/01633), thymic stroma-derived lymphopoietin, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor(GM-CSF, Australian Patent No. 588819), mast cell growth factor, stemcell growth factor (U.S. Pat. No. 6,204,363), epidermal growth factor,keratinocyte growth factor, megakaryote growth and development factor,RANTES, human fibrinogen-like 2 protein (FGL2; NCBI accession no.NM_00682; Rüegg and Pytela (1995), Gene 160:257-62) growth hormone,insulin, insulinotropin, insulin-like growth factors, parathyroidhormone, interferons including α-interferons, γ-interferon, andconsensus interferons (U.S. Pat. Nos. 4,695,623 and 4,897,471), nervegrowth factor, brain-derived neurotrophic factor, synaptotagmin-likeproteins (SLP 1-5), neurotrophin-3, glucagon, interleukins, colonystimulating factors, lymphotoxin-β, leukemia inhibitory factor, andoncostatin-M. Descriptions of proteins that can be produced according tothe inventive methods may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research, all volumes (Aggarwal andGutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook, Vols. 1 and 2(Thompson and Lotze eds., Academic Press, San Diego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Pat. Nos. 4,968,607, and 5,767,064,), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(U.S. Pat. Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be producedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified, including human,humanized, chimeric, multi-specific, monoclonal, polyclonal, andoligomers or antigen binding fragments thereof. Also included areproteins having an antigen binding fragment or region such as Fab, Fab′,F(ab′)₂, Fv, diabodies, Fd, dAb, maxibodies, single chain antibodymolecules, complementarity determining region (CDR) fragments, scFv,diabodies, triabodies, tetrabodies and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33,CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-10, IL-2,IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF,TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No. 6,235,883) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC1, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, HER-3, a tumor-associatedglycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes thatare present in elevated levels in the sera of patients with colon and/orpancreatic cancer, cancer-associated epitopes or proteins expressed onbreast, colon, squamous cell, prostate, pancreatic, lung, and/or kidneycancer cells and/or on melanoma, glioma, or neuroblastoma cells, thenecrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4,B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphlycoccus aureus. Specific examples of known antibodieswhich can be produced using the methods of the invention include but arenot limited to adalimumab, bevacizumab, infliximab, abciximab,alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

The invention can also be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Håkansson et al. (1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fc portionof an antibody such as etanercept (a p75 TNFR:Fc), and belatacept(CTLA4:Fc). Chimeric proteins and polypeptides, as well as fragments orportions, or mutants, variants, or analogs of any of the aforementionedproteins and polypeptides are also included among the suitable proteins,polypeptides and peptides that can be produced by the methods of thepresent invention.

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. The methods andtechniques described herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990). All documents, or portions of documents, cited inthis application, including but not limited to patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated by reference. What is described in an embodiment of theinvention can be combined with other embodiments of the invention.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

EXAMPLES Example 1

Extracellular ornithine levels were found to correlate with high mannoseglycoform contents. Eight CHO cell lines expressing recombinantantibodies having high mannose glycoform content ranging from <5%to >20% were chosen for this experiment (Cell line A-Cell line H). Thecells were grown in a 10 day fed-batch culture in shake flasks using twodifferent proprietary cell culture media, each containing no ornithine(Media #1 and Media #2). Spent media samples were taken on days 8, 9 and10 of the culture and were subjected to a large-scale metabolomicsanalysis. % HM was determined using an Endo-H rCE-SDS method, laterreplaced by the HILIC method described below. Relative levels ofornithine in the spent media were determined by large-scale metabolomicsanalysis in which media components were separated by liquidchromatography and detected by high-resolution spectrometry. Componentswere identified by matching their fragmentation spectra to a library ofspectra of known compounds. The relative abundance of each component wasdetermined from the peak area of its mass spectrometry signals. The highmannose glycan levels (% HM) of the secreted monoclonal antibodies fromCell lines A-H on days 8, 9 and 10 in Media #1 and Media #2 is shown inFIGS. 2A and B. FIG. 2C shows the correlation between % high mannose andextracellular ornithine levels. The correlation was determined bycomparing all 8 cell lines (represented by the squares) using data fromDay 9 samples. The results suggested a strong correlation between highmannose glycoform contents and extracellular levels of ornithine.

Next, Cell line H was grown in fed batch culture in a 3 L bioreactor.Culture duration was 12 days. Four bolus feeds of 7%, 9%, 9% and 9% weremade on days 3, 5, 7 and 9. In addition, 50% glucose solution was addeddaily starting on day 3 as required to maintain a glucose concentrationabove 2 g/L. Production bioreactors were inoculated at 15×10⁵ cells/mlafter 4 days of growth. The cells were maintained in growth medium untila production phase was initiated. Eight different process conditionswere then compared. Condition #1 acted as the control. No alterationswere made to the production feed media.

In Condition #2, betaine was supplemented in the production feed mediaat a concentration of 24 mM on day 0. No further betaine supplementswere provided. Four bolus feeds of 7%, 9%, 9% and 9% were made on days3, 5, 7 and 9. In addition, 50% glucose solution was added dailystarting on day 3 as required to maintain a glucose concentration above2 g/L.

In Conditions #3 and #4, removal of copper sulfate was tested. Coppersulfate was removed from the production base media powder. Condition #3served as a control, a copper sulfate stock solution was added to thebase media. In Condition #4, no copper sulfate was added to any media,creating a copper-deficient culture environment. For both Condition #3and #4, both were treated with the same bolus “feed” media whichcontains copper.

In Conditions #5-#8, high and low osmolality was tested. In Conditions#5 and #6, the cells were fed with 90% production batch medium, i.e.,10% less nutrients were provided, which translates to the cellsexperiencing reduced osmolarity. In Condition #6, the cell culture mediawas brought back to the control level, ˜300 mOsm, by titration withNaCl. In Condition #7 and #8 the cells were fed with 85% feed medium,with the medium in Condition #8 being brought back to the control levelby titration with NaCl.

Spent media samples were taken on days 3, 6, 8, 9 and 10 of the cultureand were subjected to a large-scale metabolomics analysis.

Again, there was a significant correlation between extracellularornithine and high mannose glycoform contents. FIG. 3A shows the percenthigh mannose glycan levels detected when Cell line H was exposed to the8 different bioreactor conditions (#1 thru #8). FIG. 3B shows thecorresponding extracellular ornithine levels. FIG. 3C shows thecorrelation between % high mannose and extracellular ornithine levels.The correlation was determined by comparing all 8 conditions(represented by the squares) using data from Day 9 samples.

Example 2

The mRNA expression levels of Arginase 1 were measured on selected daysduring a 10-day fed-batch production culture using the eight cell linesdescribed in Example 1.

The mRNA expression levels were assessed using a QuantiGene MultiplexAssay kit, (Affymetrix, Inc., Santa Clara, Calif.) according to themanufacturer's instructions.

Arginase 1, the enzyme that catalyzes the conversion of arginine toornithine, was found to be up-regulated in cell lines with higher levelsof high mannose, in a time-course dependent manner, see FIG. 4. Thissuggests that specific targeting with arginase inhibitors to block theactivity of arginase and reduce the amount of ornithine production couldbe used to lower high mannose glycan levels.

Example 3

This example demonstrates the manipulation of the high mannose glycoformcontent of recombinant glycoproteins by regulating ornithineaccumulation in the host cell expressing the recombinant glycoprotein isaddressed.

Cell Lines, Cell Culture and Media

Cell lines H was used in this study. Cells were maintained in 3 LErlenmeyer shake flasks (Corning Life Sciences, Lowell, Mass.) with 1 Lworking volume and cultivated under standard humidified condition at 36°C., 5% CO₂, and shaken at 70 rpm in an automatic CO₂ incubator (ThermoFisher Scientific, Waltham, Mass.). Cells were sub-cultured in aselective growth media containing a 500 nM concentration of methotrexate(MTX) every four days, and subsequently transferred, inoculated andcultured in a growth media for four days before being seeded in a 24wells plate for the experiments described below.

Small Scale Mock Perfusion

A modified mock perfusion in a 24 deep-well plate (Axygen, Union City,Calif.) was used to evaluate effects of spermine, arginine, ornithineand arginase concentrations on high mannose (HMN) modulation. Anarginine-free formulation of a perfusion media was used for small scalemock perfusion Experiment #3 (arginine concentration studies) accordingto the experimental design. In small scale mock perfusion Experiment #4all arginase inhibitors were added to a perfusion media. The fourarginase inhibitors, BEC Hydrochloride, DL-α, DiflouromethylornithineHydrochloride, N^(G)-Hydroxy-L-arginine Monoacetate salt andNω-Hydroxy-nor-arginine diacetate salt, were purchased from EMDMillipore Corporation (Billerica, Mass.).

Briefly, the CHO cells were seeded in the plate at the targeted densityranging from 10-20×10⁶ cells/mL with 3 mL working volume for each well.The cells were cultivated at 36° C., 5% CO₂, 85% relative humidity andshaken at 225 rpm in a 50-mm orbital diameter Kuhner incubator (KuhnerAG, Basel, Switzerland) for 3 or 4 days. Every 24 hours, the cells werecentrifuged at 200×g for 5 minutes (Beckman Coulter, Brea, Calif.) tocollect the spent media and each well was then replenished with 3 mL offresh media. The collected spent media were analyzed for titer, keymetabolites and % high mannose (% HMN) (when necessary). Cells were thenharvested and cell counts and viability were measured.

Cell Growth, Metabolites and Antibody Titer Analysis

Viable cell density and viability were determined using a Cedex cellcounter (Roche Innovative, Beilefed, Germany). Metabolites includingglucose, lactate, ammonia, glutamine, glutamate were obtained fromNovaBioprofile Flex (Nova Biomedical, Waltham, Mass.). Antibodyconcentration in the spent media was determined using a Affinity ProteinA Ultra Performance liquid chromatography (UPLC) (Waters Corporation,Milford, Mass.) assay equipped with a 50 mm×4.6 mm i.d. POROS A/20protein A column (Life Technologies, Carlsbad, Calif.). After the samplewas injected, the column was washed by Phosphate-Buffered Saline (PBS)pH=7.1 to remove CHO host cell proteins. Bounded antibodies were theneluted in acidic PBS buffer (pH=1.9) and detected by UV absorbance at280 nm to quantify antibody concentration.

HILIC Glycan Map

Different N-glycan species of antibodies were analyzed byhydrophilic-interaction liquid chromatography (HILIC). The purifiedantibodies were digested by N-glycosidase F (New England BioLabs,Ipswich, Mass.) at 37° C. for 2 hours to release the glycans. Thereleased glycans were labeled with 2-aminobenzoic acid and cleaned upusing GlycoClean S cartridges (Prozyme, Heyward, Calif.). Purifiedglycans were then desalted and reconstituted in water for assay. HILICchromatography was performed with a 100 mm×2.1 mm i.d BEH Glycan columnusing UPLC (Waters Corporation, Milford, Mass.) and the eluted glycanswere detected, identified, and qualified by a fluorescence detectorbased on different elution times of different glycans.

Small Scale Mock Perfusion Experiment #1: Spermine Concentration Study

Five different concentrations of spermine were tested in this study.Perfusion cell culture media containing 0, 7, 17, 35 and 100 μM sperminetetrahydrochloride (spermine 4HCl) were tested. The perfusion mediumcontaining 35 μM spermine acted as a control. The results from day 5samples show that as the spermine concentration was reduced, the % HMNdecreased, see FIG. 5. Titer was not affected by reduction/depletion ofspermine. Reduction of HM level was achieved through reduction inornithine level when the amount of spermine was reduced in the media. Asshown in FIG. 6, the amount of ornithine decreased with a decrease inspermine concentration.

Small Scale Mock Perfusion Experiment #2: Ornithine Concentration Study

Four different concentrations of L-ornithine monohydrochloride weretested. Perfusion cell culture media containing 14.8, 6, 0.6 and 0(control) mM L-ornithine monohydrochloride (Sigma-Aldrich, St. Louis,Mo.) were used. The results showed that as the ornithine concentrationwas increased, the % HMN increased, see FIG. 7. A second experiment wasperformed using Cell Line I in 2 L bioreactors. Cell line I expresses anIgG2 antibody and was grown under fed-batch conditions. In onebioreactor, the media received a single supplement of 0.1 g/LL-ornithine monohydrochloride on day 0 of the culture, the secondbioreactor acted as an ornithine-free control. The cultures weremaintained from 12 days in cell culture media containingsoy-hydrolysates. Bolus feed medium containing soy hydrolysates was fedon days 4 and 8.

Glycan profiling was performed by peptide mapping. The antibody wasdigested by trypsin with a method similar to described by Ren et al.(2009) Anal. Biochem. 392 12-21). Specifically, about 50-70 μg of eachantibody was denatured and reduced with 7.0 M guanidine.HCl, 6 mMdithiothreitol (DTT) in 0.2 M tris buffer (pH 7.5) at 37° C. for 30minutes. Each denatured/reduced sample was alkylated with 14 mMiodoacetic acid at 25° C. for 25 minutes, followed by quenching thereaction by adding 8 mM DTT. The reduced/alkylated antibody samples werethen exchanged into a 0.1 M tris buffer at pH 7.5 with a Piercedetergent removal spin column (Thermo Fisher Scientific Inc., Rockford,Ill.) according to manufacturer suggested protocol. The buffer-exchangedsample was incubated at 37° C. with 3.5 μg trypsin for 60 minutes.Digestion was quenched by adding 2.2 μL of 10% acetic acid. About 12-17μg of digested antibody was injected for analysis.

The digested antibody was analyzed using an Agilent 1260 HPLC systemdirectly connected to a Thermo Scientific LTQ-Orbitrap Elite massspectrometer (Thermo Fisher Scientific Inc., Rockford, Ill.).Proteolytic peptides were separated on a Waters BEH 300 C18 column(Waters Corporation, Milford, Mass.) 2.1×150 mm, 1.7μ particle at 40° C.with a flow rate of 0.2 mL/min. Mobile phase A was 0.02% TFA in waterand mobile phase B was 0.018% TFA in acetonitrile. Peptides were elutedwith a gradient of 0.5-40% B in 90 minutes, followed by column washingand re-equilibration. Mass spectrometer was set up for a full MS scan inthe orbitrap with 120,000 resolution followed by five data-dependent CIDMS/MS scans in the linear trap with dynamic exclusion. Automated dataanalysis for glycan profiling was performed using MassAnalyzer (seeZhang, (2009) Analytical Chemistry 81: 8354-8364).

The results again show that as the ornithine concentration is increasedthe % HMN increased, see FIG. 8.

Small Scale Mock Perfusion Experiment #3: Arginine Concentration Study

Five different concentrations of arginine were tested in this study.Perfusion cell culture media containing 3.686, 1.38, 0.92 and 0.46 g/Larginine were tested. The perfusion medium containing 1.843 g/L argininewas used as a control. The results show that as the arginineconcentration is increased the % HM increased, see FIG. 9.

Small Scale Mock Perfusion Experiment #4: Arginase Inhibitor Studies

Two series of arginase inhibitor experiments were conducted. In thefirst series of experiments, four commercially available arginaseinhibitors BEC Hydrochloride, DL-α, DiflouromethylornithineHydrochloride, N^(G)-Hydroxy-L-arginine Monoacetate salt andNω-Hydroxy-nor-arginine diacetate salt were added to the cell culturesat three different concentrations, 1, 10 and 20 μM. The control wasinhibitor free. From this experiment it was concluded the inhibitors BECand DL-α were most effective in decreasing % HM (FIG. 10).

A second series experiments were conducted using the BEC and DL-αinhibitors. The BEC inhibitor was tested at 0 (control), 10 μM and 0.5mM concentrations in perfusion cell culture media. The DL-α inhibitorswas tested at 0 (control), 10 μM, 1.0 mM and 2.0 mM concentrations inperfusion culture media. It was demonstrated that % HM was decreased asboth inhibitor concentrations increased, see FIG. 11.

What is claimed is:
 1. A method of increasing high mannose glycoformcontent of a recombinant protein comprising culturing a host cellexpressing the recombinant protein in a cell culture comprisingornithine or arginine, wherein ornithine production in the host cell isincreased when compared to the host cell expressing the recombinantprotein is cultured in a cell culture lacking ornithine or arginine, andthe recombinant protein has an increased high mannose glycoform contentthan when the recombinant protein is expressed in the cell culturelacking ornithine or arginine.
 2. The method of claim 1, whereinornithine accumulation in the host cell is increased by the addition ofat least 0.6 mM ornithine to the cell culture.
 3. The method of claim 1,wherein the concentration of ornithine is selected from the groupconsisting of 0.6 to 14.8 mM; 6 to 14.8 mM; 0.6 mM, 6 mM, and 14.8 mM.4. The method of claim 1, wherein ornithine accumulation in the hostcell is regulated by the addition of at least 8.7 mM arginine to cellculture medium.
 5. The method of claim 1, wherein the concentration ofarginine is selected from the group consisting of 8.7 mM to 17.5 mM; 8.7mM; and 17.5 mM.
 6. The method of claim 1, wherein the host cellexpressing the recombinant protein is cultured in a batch culture,fed-batch culture, perfusion culture, or combinations thereof.
 7. Themethod of claim 1, wherein the host cell expressing the recombinantprotein is cultured in a bioreactor.
 8. The method of claim 7, whereinthe bioreactor has a capacity selected from the group consisting of atleast 500 L; 500 L to 2000 L; and 1000 L to 2000 L.
 9. The method ofclaim 1, wherein the host cell expressing the recombinant protein iscultured in a serum-free cell culture medium.
 10. The method of claim 9,wherein the serum-free culture medium is a perfusion cell culturemedium.
 11. The method of claim 1, wherein the host cells are mammaliancells.
 12. The method of claim 1, wherein the host cells are ChineseHamster Ovary (CHO) cells.
 13. The method of claim 1, wherein therecombinant protein is a glycoprotein.
 14. The method of claim 1,wherein the recombinant protein is selected from the group consisting ofa human antibody, a humanized antibody, a chimeric antibody, arecombinant fusion protein, or a cytokine.
 15. The method of claim 1,further comprising a step of harvesting the recombinant protein producedby the host cell.
 16. The method of claim 1, wherein the recombinantprotein produced by the host cell is purified and formulated into apharmaceutically acceptable formulation.